Modification of rna, producing an increased transcript stability and translation efficiency

ABSTRACT

It was the object of the present invention to provide RNA with increased stability and translation efficiency and means for obtaining such RNA. It should be possible to obtain increased grades of expression by using said RNA in gene therapy approaches.

RELATED APPLICATIONS

This application is a U.S. National Stage Application submitted under 35U.S.C. 371 of International Application Serial Number PCT/EP2006/009448,filed on Sep. 28, 2006, which claims priority to German ApplicationSerial No. 102005046490.4, filed on Sep. 28, 2005. The entire teachingsof the above-referenced Application are incorporated herein byreference.

BACKGROUND

Conventional vaccines, including attenuated or inactivated pathogens,are effective in many areas but nevertheless do not impart effectiveprotective immunity to some infectious pathogens and tumors. Thisrequires vaccines which are effective, versatile, ready andcost-effective to produce and easy to store.

After direct intramuscular injection of plasmid DNA had been shown toresult in prolonged expression of the coded genes on the cell surface(Wolff et al., 1990), DNA-based vaccines were regarded as a newpromising immunization strategy. This provided an important incentivefor developing vaccines based on nucleic acids. Initially, DNA-basedvaccines to infectious pathogens were tested (Cox et al., 1993; Davis etal., 1993; Ulmer et al., 1993; Wang et al., 1993) but were soon howeverresearched in more detail also in gene therapy against tumors in orderto induce specific antitumor immunity (Conry et al., 1994; Conry et al.,1995a; Spooner et al., 1995; Wang et al., 1995). This strategy of tumorimmunization has a number of important advantages. Vaccines based onnucleic acids are easy to prepare and relatively inexpensive. They maymoreover be amplified from a small number of cells.

DNA is more stable than RNA but carries some potential safety risks suchas the induction of anti-DNA antibodies (Gilkeson et al., 1995) andintegration of the transgen into the host genome. This may inactivatecellular genes, cause uncontrollable long term expression of saidtransgen or oncogenesis and is therefore usually not applicable totumor-associated antigens with oncogenic potential, such as, forexample, erb-B2 (Bargmann et al., 1986) and p53 (Greenblatt et al.,1994). The use of RNA offers an attractive alternative in order tocircumvent these potential risks.

The advantages of using RNA as a kind of reversible gene therapy includetransient expression and a non-transforming character. The RNA does notneed to enter the nucleus in order to be expressed transgenically andmoreover cannot integrate into the host genome, thereby eliminating therisk of oncogenesis. As with DNA (Condon et al., 1996; Tang et al.,1992), injection of RNA can also induce both the cellular and humoralimmune responses in vivo (Hoerr et al., 2000; Ying et al., 1999).

The immune therapy with in vitro-transcribed RNA (IVT-RNA) makes use oftwo different strategies both of which have been successively tested invarious animal models. Either RNA is directly injected via differentroutes of immunization (Hoerr et al., 2000) or dendritic cells (DCs) aretransfected with in vitro-transcribed RNA by means of lipofection orelectroporation and administered thereafter (Heiser et al., 2000).Recently published studies demonstrated that immunization withRNA-transfected DCs induces antigen-specific cytotoxic T lymphocytes(CTL) in vitro and in vivo (Su et al., 2003; Heiser et al., 2002). Afactor of central importance for optimal induction of the Tcell-mediated immune responses is inter alia the dose, i.e. density ofantigen presentation on the DCs. It has been attempted to stabilizeIVT-RNA by various modifications in order to achieve prolongedexpression of transferred IVT-RNA and thereby to increase antigenpresentation on DCs. A basic requirement for translation is the presenceof a 3′ poly(A) sequence, with the translation efficiency correlatingwith the length of poly(A) (Preiss and Hentze, 1998). The 5′ cap and 3′poly(A) sequence synergistically activate translation in vivo (Gallie,1991). Untranslated regions (UTRs) of globin genes are other knownelements which can contribute to stabilizing RNA and increasingtranslation efficiency (Malone et al., 1989).

Some IVT vectors are known in the literature which are utilized in astandardized manner as template for in vitro transcription and whichhave been genetically modified in such a way that stabilized RNAtranscripts are produced. Protocols currently described in theliterature (Conry et al., 1995b; Teufel et al., 2005; Strong et al.,1997; Carralot et al., 2004; Boczkowski et al., 2000) are based on aplasmid vector with the following structure: a 5′ RNA polymerasepromoter enabling RNA transcription, followed by a gene of interestwhich is flanked either 3′ and/or 5′ by untranslated regions (UTR), anda 3′ polyadenyl cassette containing 50-70 A nucleotides. Prior to invitro transcription, the circular plasmid is linearized downstream ofthe polyadenyl cassette by type II restriction enzymes (recognitionsequence corresponds to cleavage site). The polyadenyl cassette thuscorresponds to the later poly(A) sequence in the transcript. As a resultof this procedure, some nucleotides remain as part of the enzymecleavage site after linearization and extend or mask the poly(A)sequence at the 3′ end. It is not clear, whether this nonphysiologicaloverhang affects the amount of protein produced intracellularly fromsuch a construct.

RNA therefore seems to be particularly suitable for clinicalapplications. However, the utilization of RNA in gene therapy is greatlyrestricted especially by the short half life of RNA, in particular inthe cytoplasma, resulting in low protein expression.

BRIEF SUMMARY OF THE INVENTION

It was the object of the present invention to provide RNA with increasedstability and translation efficiency and means for obtaining such RNA.It should be possible to obtain increased grades of expression by usingsaid RNA in gene therapy approaches.

This object is achieved according to the invention by the subject matterof the claims.

The present invention relates to stabilization of RNA, in particularmRNA, and an increase in mRNA translation. The present inventionparticularly relates to three modifications of RNA, in particular invitro-transcribed RNA, resulting in increased transcript stability andtranslation efficiency.

According to the invention, RNA having an open-ended poly(A) sequencewas found to be translated more efficiently than RNA having a poly(A)sequence with a masked terminus. It was found that a long poly(A)sequence, in particular of about 120 bp, results in optimal RNAtranscript stability and translation efficiency. The invention alsodemonstrated that a double 3′-untranslated region (UTR), in particularof the human beta-globin gene, in an RNA molecule improves translationefficiency in a way which clearly exceeds the total effect to beexpected using two individual UTRs. A combination of the above-describedmodifications was found according to the invention to have a synergisticinfluence on the stabilization of RNA and the increase in translation.

Using quantitative RT-PCR and eGFP variants for measuring transcriptquantities and protein yield, the invention demonstrated that the RNAmodifications according to the invention independently enhance RNAstability and translation efficiency in the transfection of dendriticcells (DCs). Thus it was possible to increase the density ofantigen-specific peptide/MHC complexes on the transfected cells andtheir ability to stimulate and expand antigen-specific CD4⁺ and CD8⁺ Tcells. The invention therefore relates to a strategy for optimizingRNA-transfected DC vaccines by using RNA which has been modified by theRNA modifications described according to the invention.

According to the invention, modification, and thereby stabilizationand/or increase in translation efficiency, of RNA is preferably achievedby genetically modifying expression vectors which preferably serve astemplate for RNA transcription in vitro.

Vectors of this kind are intended to allow in particular transcriptionof RNA with a poly(A) sequence which preferably has an open end in saidRNA, i.e. no nucleotides other than A nucleotides flank said poly(A)sequence at its 3′ end. An open-ended poly(A) sequence in the RNA can beachieved by introducing a type IIS restriction cleavage site into anexpression vector which allows RNA to be transcribed under the controlof a 5′ RNA polymerase promoter and which contains a polyadenyl cassette(poly(A) sequence), wherein the recognition sequence is located 3′ ofthe poly(A) sequence, while the cleavage site is located upstream andthus within the poly(A) sequence. Restriction cleavage at the type IISrestriction cleavage site enables a plasmid to be linearized within thepoly(A) sequence (FIG. 2 ). The linearized plasmid can then be used astemplate for in vitro transcription, the resulting transcript ending inan unmasked poly(A) sequence.

Furthermore or alternatively, a modification, and thereby stabilizationand/or increase in translation efficiency, of RNA can be achievedaccording to the invention by genetically modifying expression vectorsin such a way that they allow transcription of RNA with two or more3′-untranslated regions at its 3′ end, and preferably between thesequence coding for a peptide or protein (open reading frame) and thepoly(A) sequence.

In one aspect, the invention relates to a nucleic acid moleculecomprising in the 5′ → 3′ direction of transcription: (a) a promoter;(b) a transcribable nucleic acid sequence or a nucleic acid sequence forintroducing a transcribable nucleic acid sequence; (c-1) a first nucleicacid sequence which corresponds to the 3′-untranslated region of a geneor is derived therefrom; and (c-2) a second nucleic acid sequence whichcorresponds to the 3′-untranslated region of a gene or is derivedtherefrom.

In one embodiment, the nucleic acid molecule according to the inventionfurther comprises (c-3) at least one further nucleic acid sequence whichcorresponds to the 3′-untranslated region of a gene or is derivedtherefrom.

In the nucleic acid molecule according to the invention, the nucleicacid sequences (b), (c-1), (c-2) and, where appropriate, (c-3) under thecontrol of the promoter (a) can preferably be transcribed to give acommon transcript in which the nucleic acid sequences transcribed fromthe nucleic acid sequences (c-1) and/or (c-2) and/or, where appropriate,(c-3) are preferably active so as to increase the translation efficiencyand/or the stability of the nucleic acid sequence transcribed from thetranscribable nucleic acid sequence (b).

The nucleic acid sequences (c-1), (c-2) and, where appropriate, (c-3)may be identical or different.

In one embodiment, the nucleic acid molecule further comprises (d) anucleic acid sequence which, when transcribed under the control of thepromoter (a), codes for a nucleotide sequence of at least 20 consecutiveA nucleotides in the transcript.

The nucleic acid sequences (b), (c-1), (c-2), where appropriate (c-3),and (d) under the control of the promoter (a) can preferably betranscribed to give a common transcript in which the nucleic acidsequences transcribed from the nucleic acid sequences (c-1) and/or (c-2)and/or, where appropriate, (c-3) and/or (d) are preferably active so asto increase the translation efficiency and/or the stability of thenucleic acid sequence transcribed from the transcribable nucleic acidsequence (b).

In particular embodiments, the nucleic acid sequence (d), whentranscribed under the control of the promoter (a), codes for anucleotide sequence of at least 40, preferably at least 80, preferablyat least 100, and in particular about 120, consecutive A nucleotides inthe transcript. The nucleic acid sequence (d), when transcribed underthe control of the promoter (a), preferably codes for a nucleotidesequence of up to 500, preferably up to 400, preferably up to 300,preferably up to 200, and in particular up to 150, consecutive Anucleotides in the transcript.

In one embodiment, the nucleic acid molecule is characterized in that itcan be cleaved, preferably enzymatically or in another biochemical way,within the nucleic acid sequence (d) in such a way that said cleavageresults in a nucleic acid molecule which comprises, in the 5′ → 3′direction of transcription, the promoter (a), the nucleic acid sequence(b), the nucleic acid sequences (c-1), (c-2) and, where appropriate,(c-3), and at least a part of the nucleic acid sequence (d), wherein theat least a part of the nucleic acid sequence (d), when transcribed underthe control of the promoter (a), codes for a nucleotide sequence of atleast 20 consecutive A nucleotides in the transcript and wherein in thetranscript the 3′-terminal nucleotide is an A nucleotide of saidnucleotide sequence of at least 20 consecutive A nucleotides.

Preferably, after cleavage, said nucleic acid molecule, at the end ofthe strand that serves as template for the nucleotide sequence of atleast 20 consecutive A nucleotides, has a T nucleotide which is part ofthe nucleotide sequence which serves as template for said nucleotidesequence of at least 20 consecutive A nucleotides in the transcript.

In particular embodiments, the at least part of the nucleic acidsequence (d), when transcribed under the control of the promoter (a),codes for a nucleotide sequence of at least 40, preferably at least 80,preferably at least 100, and in particular about 120, consecutive Anucleotides in the transcript. The nucleic acid sequence (d), whentranscribed under the control of the promoter (a), preferably codes fora nucleotide sequence of up to 500, preferably up to 400, preferably upto 300, preferably up to 200, and in particular up to 150, consecutive Anucleotides in the transcript.

The nucleic acid molecule according to the invention is preferably aclosed circular molecule prior to cleavage and a linear molecule aftercleavage.

Preferably, cleavage is carried out with the aid of a restrictioncleavage site which is preferably a restriction cleavage site for a typeIIS restriction endonuclease.

In one embodiment, the recognition sequence for the type IIS restrictionendonuclease is located 5-26 base pairs, preferably 24-26 base pairs,downstream of the 3′ end of the nucleic acid sequence (d).

In preferred embodiments, the nucleic acid sequences (c-1), (c-2) and,where appropriate, (c-3) are independently of one another derived from agene selected from the group consisting of globin genes such asalpha2-globin, alpha1-globin, beta-globin and growth hormone, preferablyhuman beta-globin, and correspond, in a particularly preferredembodiment, to the nucleic acid sequence according to SEQ ID No. 1 ofthe sequence listing or to a nucleic acid sequence derived therefrom.

In a further aspect, the invention relates to a nucleic acid moleculecomprising in the 5′ → 3′ direction of transcription: (a) a promoter;(b) a transcribable nucleic acid sequence or a nucleic acid sequence forintroducing a transcribable nucleic acid sequence; and (c) a nucleicacid sequence which, when transcribed under the control of the promoter(a), codes for a nucleotide sequence of at least 20 consecutive Anucleotides in the transcript.

The nucleic acid sequences (b) and (c) under the control of the promoter(a) can preferably be transcribed to give a common transcript in whichthe nucleic acid sequence transcribed from the nucleic acid sequence (c)is preferably active so as to increase the translation efficiency and/orthe stability of the nucleic acid . sequence transcribed from thetranscribable nucleic acid sequence (b).

In particular embodiments, the nucleic acid sequence (c), whentranscribed under the control of the promoter (a), codes for anucleotide sequence of at least 40, preferably at least 80, preferablyat least 100, and in particular about 120, consecutive A nucleotides inthe transcript. The nucleic acid sequence (c), when transcribed underthe control of the promoter (a), preferably codes for a nucleotidesequence of up to 500, preferably up to 400, preferably up to 300,preferably up to 200, and in particular up to 150, consecutive Anucleotides in the transcript.

In one embodiment, the nucleic acid molecule can be cleaved, preferablyenzymatically or in another biochemical way, within the nucleic acidsequence (c) in such . a way that said cleavage results in a nucleicacid molecule which comprises, in the 5′ → 3′ direction oftranscription, the promoter (a), the nucleic acid sequence (b), and atleast a part of the nucleic acid sequence (c), wherein the at least apart of the nucleic acid sequence (c), when transcribed under thecontrol of the promoter (a), codes for a nucleotide sequence of at least20 consecutive A nucleotides in the transcript and wherein in thetranscript the 3′-terminal nucleotide is an A nucleotide of saidnucleotide sequence of at least 20 consecutive A nucleotides.

Preferably, after cleavage, the nucleic acid molecule, at the end of thestrand that serves as template for the nucleotide sequence of at least20 consecutive A nucleotides, has a T nucleotide which is part of thenucleotide sequence which serves as template for the nucleotide sequenceof at least 20 consecutive A nucleotides in the transcript.

In particular embodiments, the at least a part of the nucleic acidsequence (c), when transcribed under the control of the promoter (a),codes for a nucleotide sequence of at least 40, preferably at least 80,preferably at least 100, and in particular about 120, consecutive Anucleotides in the transcript. The nucleic acid sequence (c), whentranscribed under the control of the promoter (a), preferably codes fora nucleotide sequence of up to 500, preferably up to 400, preferably upto 300, preferably up to 200, and in particular up to 150, consecutive Anucleotides in the transcript.

The nucleic acid molecule is preferably a closed circular molecule priorto cleavage and a linear molecule after cleavage.

Preferably, cleavage is carried out with the aid of a restrictioncleavage site which is preferably a restriction cleavage site for a typeIIS restriction endonuclease.

In one embodiment, the recognition sequence for the type IIS restrictionendonuclease is located 5-26 base pairs, preferably 24-26 base pairs,downstream of the 3′ end of the nucleic acid sequence (c).

In one nucleic acid molecule according to the invention, thetranscribable nucleic acid sequence preferably comprises a nucleic acidsequence coding for a peptide or protein and the nucleic acid sequencefor introducing a transcribable nucleic acid sequence is preferably amultiple cloning site.

A nucleic acid molecule according to the invention may further compriseone or more members selected from the group consisting of: (i) areporter gene; (ii) a selectable marker; and (iii) an origin ofreplication.

In one embodiment, a nucleic acid molecule according to the invention isin a closed circular conformation and preferably suitable for in vitrotranscription of RNA, in particular mRNA, in particular afterlinearization.

In further aspects, the invention relates to a nucleic acid moleculeobtainable by linearization of an above-described nucleic acid molecule,preferably by cleavage within the nucleic acid sequence which codes fora nucleotide sequence of at least 20 consecutive A nucleotides, and toRNA obtainable by transcription, preferably in vitro transcription, withabove-described nucleic acid molecules under the control of the promoter(a).

In a further aspect, the invention relates to a method of transcribingin vitro a selected RNA molecule in order to increase its stabilityand/or translation efficiency, comprising: (i) coupling a first nucleicacid sequence (b-1) which corresponds to the 3′-untranslated region of agene or is derived therefrom at the 3′ end of a nucleic acid sequence(a) which can be transcribed to give said RNA molecule, (ii) coupling asecond nucleic acid sequence (b-2) which corresponds to the3′-untranslated region of a gene or is derived therefrom at the 3′ endof said first nucleic acid sequence (b-1), and (iii) transcribing invitro the nucleic acid obtained.

In a further aspect, the invention relates to a method of translating aselected mRNA molecule in order to increase expression thereof,comprising: (i) coupling a first nucleic acid sequence (b-1) whichcorresponds to the 3′-untranslated region of a gene or is derivedtherefrom at the 3′ end of a nucleic acid sequence (a) which can betranscribed to give said mRNA molecule, (ii) coupling a second nucleicacid sequence (b-2) which corresponds to the 3′-untranslated region of agene or is derived therefrom at the 3′ end of said first nucleic acidsequence (b-1), and (iii) translating the mRNA which is obtainable bytranscribing the nucleic acid obtained. Transcription is preferablycarried out in vitro.

According to the invention, the term “coupling a nucleic acid sequenceat the 3′ end of a nucleic acid sequence” relates to a covalent linkageof the two nucleic acid sequences in such a way that the first nucleicacid sequence is downstream of the second nucleic acid sequence and maybe separated from the latter by additional nucleic acid sequences.

In one embodiment, the methods according to the invention furthercomprise coupling at least one further nucleic acid sequence (b-3) whichcorresponds to the 3′-untranslated region of a gene or is derivedtherefrom at the 3′ end of the second nucleic acid sequence (b-2).

The nucleic acid sequences (a), (b-1), (b-2) and, where appropriate,(b-3) can preferably be transcribed to give a common transcript in whichthe nucleic acid sequences transcribed from the nucleic acid sequences(b-1) and/or (b-2) and/or, where appropriate, (b-3) are preferablyactive so as to increase the translation efficiency and/or the stabilityof the nucleic acid sequence transcribed by the nucleic acid sequence(a).

In a further embodiment, the methods according to the invention furthercomprise coupling a nucleic acid sequence (c) which, when transcribed,codes for a nucleotide sequence of at least 20 consecutive Anucleotides, at the 3′ end of the nucleic acid sequence (b-2) or, whereappropriate, of the nucleic acid sequence (b-3).

The nucleic acid sequences (a), (b-1), (b-2) and, where appropriate,(b-3), and (c) can preferably be transcribed to give a common transcriptin which the nucleic acid sequences transcribed from the nucleic acidsequences (b-1) and/or (b-2) and/or, where appropriate, (b-3), and/or(c) are preferably active so as to increase the translation efficiencyand/or the stability of the nucleic acid sequence transcribed from thenucleic acid sequence (a).

In particular embodiments, the nucleic acid sequence (c), whentranscribed, codes for a nucleotide sequence of at least 40, preferablyat least 80, preferably at least 100, and in particular about 120,consecutive A nucleotides in the transcript. The nucleic acid sequence(c), when transcribed, preferably codes for a nucleotide sequence of upto 500, preferably up to 400, preferably up to 300, preferably up to200, and in particular up to 150, consecutive A nucleotides in thetranscript.

In particular embodiments, the methods according to the inventionfurther comprise, prior to transcription of the nucleic acid obtained,cleavage within the nucleic acid sequence (c) in such a way thattranscription of the nucleic acid obtained in this way generates atranscript which has the nucleic acid sequences transcribed from thenucleic acid sequences (a), (b-1), (b-2) and, where appropriate, (b-3)and a 3′-terminal nucleotide sequence of at least 20 consecutive Anucleotides, wherein the 3′-terminal nucleotide of said transcript is anA nucleotide of the nucleotide sequence of at least 20 consecutive Anucleotides.

In particular embodiments, the transcript has at its 3′ end a nucleotidesequence of at least 40, preferably at least 80, preferably at least100, and in particular about 120, consecutive A nucleotides. Thetranscript preferably has at its 3′ end a nucleotide sequence of up to500, preferably up to 400, preferably up to 300, preferably up to 200,and in particular up to 150, consecutive A nucleotides.

In preferred embodiments, the nucleic acid sequences (b-1), (b-2) and,where appropriate, (b-3) are independently of one another derived from agene selected from the group consisting of globin genes such asalpha2-globin, alphal-globin, beta-globin and growth hormone, preferablyhuman beta-globin, and correspond, in a particularly preferredembodiment, to the nucleic acid sequence according to SEQ ID No. 1 ofthe sequence listing or to a nucleic acid sequence derived therefrom.

In a further aspect, the invention relates to a method of transcribingin vitro a selected RNA molecule in order to increase its stabilityand/or translation efficiency, comprising: (i) coupling a nucleic acidsequence (b) which, when transcribed, codes for a nucleotide sequence ofat least 20 consecutive A nucleotides, at the 3′ end of a nucleic acidsequence (a) which can be transcribed to give said RNA molecule, and(ii) transcribing in vitro the nucleic acid obtained.

In a further aspect, the invention relates to a method of translating aselected mRNA molecule in order to increase expression thereof,comprising: (i) coupling a nucleic acid sequence (b) which, whentranscribed, codes for a nucleotide sequence of at least 20 consecutiveA nucleotides, at the 3′ end of a nucleic acid sequence (a) which can betranscribed to give said mRNA molecule, and (ii) translating the mRNAwhich is obtainable by transcribing the nucleic acid obtained.Transcription is preferably carried out in vitro.

The nucleic acid sequences (a) and (b) can preferably be transcribed togive a common transcript in which the nucleic acid sequence transcribedfrom the nucleic acid sequence (b) is preferably active so as toincrease the translation efficiency and/or the stability of the nucleicacid sequence transcribed from the nucleic acid sequence (a).

In particular embodiments, the nucleic acid sequence (b), whentranscribed, codes for a nucleotide sequence of at least 40, preferablyat least 80, preferably at least 100, and in particular about 120,consecutive A nucleotides in the transcript. The nucleic acid sequence(b), when transcribed, preferably codes for a nucleotide sequence of upto 500, preferably up to 400, preferably up to 300, preferably up to200, and in particular up to 150, consecutive A nucleotides in thetranscript.

In particular embodiments, the methods according to the inventionfurther comprise, prior to transcription of the nucleic acid obtained,cleavage within the nucleic acid sequence (b) in such a way thattranscription of the nucleic acid obtained in this way generates atranscript which has the nucleic acid sequences transcribed from thenucleic acid sequence (a) and a 3′-terminal nucleotide sequence of atleast 20 consecutive A nucleotides, wherein the 3′-terminal nucleotideof said transcript is an A nucleotide of the nucleotide sequence of atleast 20 consecutive A nucleotides.

In particular embodiments, the transcript has at least 40, preferably atleast 80, preferably at least 100, and in particular about 120,consecutive A nucleotides at its 3′ end. The transcript preferably hasup to 500, preferably up to 400, preferably up to 300, preferably up to200, and in particular up to 150, consecutive A nucleotides at its 3′end.

In all aspects of the methods according to the invention, cleavage ispreferably carried out with the aid of a restriction cleavage site whichis preferably a restriction cleavage site for a type IIS restrictionendonuclease.

In one embodiment, the recognition sequence for the type IIS restrictionendonuclease is 5-26 base pairs, preferably 24-26 base pairs, downstreamof the 3′ end of the nucleic acid sequence which, when transcribed,codes for a nucleotide sequence of at least 20 consecutive Anucleotides.

The invention also relates to RNA obtainable by the methods according tothe invention of transcribing in vitro a selected RNA molecule. The RNApreparation obtainable by the methods according to the invention oftranscribing in vitro a selected RNA molecule from a nucleic acidmolecule according to the invention as template is preferablyhomogeneous or essentially homogeneous with regard to the length of thepoly(A) sequence of the RNA, i.e. the length of the poly (A) sequence inmore than 90%, preferably more than 95%, preferably more than 98% or99%, of the RNA molecules in the preparation differs by no more than 10,preferably no more than 5, 4, 3, 2 or 1, A nucleotides.

The invention may be utilized, for example, for increasing expression ofrecombinant proteins in cellular transcription and expression. Morespecifically, it is possible, when producing recombinant proteins, tointroduce the modifications described according to the invention and acombination thereof into expression vectors and utilize them for thepurpose of increasing transcription of recombinant. nucleic acids andexpression of recombinant proteins in cell-based systems. This includes,for example, the preparation of recombinant antibodies, hormones,cytokines, enzymes, and the like. This allows inter alia productioncosts to be reduced.

It is also possible to utilize the modifications described according tothe invention and a combination thereof for gene therapy applications.Said modifications may be introduced into gene therapy vectors andthereby utilized for increasing expression of a transgen. To this end,any nucleic acid (DNA/RNA)-based vector systems (for example plasmids,adenoviruses, poxvirus vectors, influenza virus vectors, alphavirusvectors, and the like) may be used. Cells can be transfected with thesevectors in vitro, for example in lymphocytes or dendritic cells, or elsein vivo by direct administration.

It is further possible for the modifications described according to theinvention and a combination thereof to increase the stability and/orexpression efficiency of ribonucleic acids and thereby the amount of thepeptides or proteins encoded by said ribonucleic acids. Codingribonucleic acids may be employed, for example, for transient expressionof genes, with possible fields of application being RNA-based vaccineswhich are transfected into cells in vitro or administered directly invivo, transient expression of functional recombinant proteins in vitro,for example in order to initiate differentiation processes in cells orto study functions of proteins, and transient expression of functionalrecombinant proteins such as erythropoietin, hormones, coagulationinhibitors, etc., in vivo, in particular as pharmaceuticals.

RNA, in particular in vitro-transcribed RNA, modified by themodifications described according to the invention, may be used inparticular for transfecting antigen-presenting cells and thus as a toolfor delivering the antigen to be presented and for loadingantigen-presenting cells, with said antigen to be presentedcorresponding to the peptide or protein expressed from said RNA or beingderived therefrom, in particular by way of intracellular processing suchas cleavage, i.e. the antigen to be presented is, for example, afragment of the peptide or protein expressed from the RNA. Suchantigen-presenting cells may be used for stimulating T cells, inparticular CD4⁺ and/or CD8⁺ T cells.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1: Basic Vectors Used According tothe Invention for Further Cloning

The vectors allow RNA transcription under the control of an RNApolymerase 5′ promoter and contain a polyadenyl cassette.

FIG. 2: Linearization of Vectors by Type II Restriction Enzymes (e.g.SpeI) in Comparison with Type IIS Restriction Enzymes (e.g. SapI)

By introducing a type IIS restriction cleavage site whose recognitionsequence is located 3′ of the poly(A) sequence, while the cleavage siteis 24-26 bp upstream and thus located within the poly(A) sequence, it ispossible to linearize a plasmid within the poly(A) sequence.

FIG. 3: Vectors Prepared According to the Invention as Template for InVitro Transcription

In order to study the effects of RNA modifications according to theinvention on the level and duration of expression, a number of vectorswere prepared which subsequently served as template for in vitrotranscription, a. Vectors with masked versus unmasked poly(A) sequence;b. Vectors -with poly(A) sequences of different length; c. Vectors with3′-untranslated region of human beta-globin; d. SIINFEKL and pp65vectors; Cap - 5′-capping; eGFP - GFP reporter gene; 3′βg -3′-untranslated region of β-globin; A(x) - x refers to the number of Anucleotides in the poly (A) sequence.

FIG. 4: Determination of the Maturation State Of Immature Versus MatureDendritic Cells by Way of the Surface Markers Indicated

The effect of the RNA modifications according to the invention wastested in human dendritic cells (DCs), with an immunogenic stimulustriggering a DC maturation process. The DCs were stained with anti-CD80,anti-CD83, anti-CD86 and anti-HLA-DR antibodies which recognize specificDC maturation markers, and analyzed by flow cytometry.

FIG. 5: Influence of Free Versus Masked poly(A) Sequence on TranslationEfficiency and Transcript Stability

a. Influence of free versus masked poly(A) sequence on the translationefficiency of eGFP RNA in K562 cells and dendritic cells by way ofdetermining the mean fluorescence intensity [MFI] in FACS-Kalibur; b.Influence of free versus masked poly(A) sequence on the transcriptstability of eGFP RNA in immature dendritic cells after 48 h. In boththe tumor cell line and in immature DCs, RNA with an open-ended poly(A)sequence is translated more efficiently and over a longer period thanRNA with a masked-end poly(A) sequence. The translation efficiency foran unmasked-end poly(A) sequence in DCs is increased by a factor of 1.5,with poly(A) sequences of equal length. An open-ended poly(A) sequencemoreover results in higher RNA stability.

FIG. 6: Influence of poly(A) Sequence Length On Translation Efficiencyand Transcript Stability

a. Influence of poly(A) sequence length on the translation efficiency ofeGFP RNA in K562 cells and dendritic cells; b. Influence of poly(A)sequence length on the translation efficiency of d2eGFP RNA in K562cells and dendritic cells; c. Influence of poly(A) sequence length onthe transcript stability of eGFP RNA in K562 cells 48 h afterelectroporation. Extending the poly(A) sequence up to 120 A nucleotidesincreases the stability and translation of the transcript. An extensionin excess of this has no positive effect. Extending the poly(A) sequencefrom 51 to 120 A nucleotides produces a 1.5 to 2-fold increase intranslation efficiency. This effect is also reflected in RNA stability.

FIG. 7: Influence of a 3′-untranslated Region of Human Beta-Globin(BgUTR) on Translation Efficiency in Immature and Mature DCs

Introducing a 3′-untranslated region of human beta-globin results inincreasing expression of the RNA transcript. A double 3′-untranslatedregion of human beta-globin enhances the level of expression after 24 h,with said level markedly exceeding the combined effect of two individual3′-untranslated regions of human beta-globin.

FIG. 8: Effect of the Combined Modifications According to the Inventionon Translation Efficiency in Immature and Mature DCs

The translation efficiency of eGFP in immature and mature DCs can beincreased by a factor of more than five by combining the RNA transcriptmodifications described according to the invention.

FIG. 9: Effect of the Combined Modifications According to the Inventionon the Presentation of Peptides by MHC Molecules on EL4 cells

Using the RNA constructs modified according to the invention results inenhanced presentation of peptide-MHC complexes on the cell surface, dueto increased translation efficiency. In the IVT vectors described, eGFPwas replaced with the OVA257-264 epitope (SIINFEKL) and EL4 cells(murine, T cell lymphoma) were used as target cells for transfection.

FIG. 10: Increase of Antigen-Specific Peptide/MHC Complexes by Using IVTRNA Constructs Stabilized According to the Invention

Cells were electroporated with Sec-SIINFEKL-A67-ACUAG RNA orSec-SIINFEKL-2BgUTR-A120 RNA (EL4 cells: 10 pmol, 50 pmol; C57B1/J6immature BMDCs in triplicates: 150 pmol). Electroporation with bufferonly was used as control. Cells were stained with 25D1.16 antibodieswith regard to SIINFEKL/K^(b) complexes. SIINFEKL peptide concentrationswere calculated from the average fluorescence values of living cells,using a peptide titration as standard curve. BMDC data are shown asaverages of three experiments ± SEM.

FIG. 11: Effect of IVT RNA Constructs Stabilized According to theInvention on T cell Stimulation In Vivo and In Vitro

(A) Improved in vivo T cell expansion by using stabilized IVT RNAconstructs. 1 × 10⁵ TCR-transgenic CD8⁺ OT-I cells were adoptivelytransferred into C57B1/J6 mice. BMDCs of C57B1/J6 mice were transfectedwith 50 pmol of RNA (Sec-SIINFEKL-A67-ACUAG, Sec-SIINFEKL-2BgUTR-A120 orcontrol RNA), matured with poly(I:C) (50 µg/ml) for 16 h and injectedi.p. one day after T cell transfer (n=3). Peripheral blood was taken onday 4 and stained for SIINFEKL tetramer-positive CD8⁺ T cells. Dot blotsdepict CD8⁺ T cells, and the numbers indicated represent the percentageof tetramer-positive CD8⁺ T cells.

(B) Improved in vitro expansion of human T cells containing stabilizedIVT RNA constructs. CD8⁺ and CD4⁺ lymphocytes from HCMV-seropositivehealthy donors were cocultured with autologous DCs which had beentransfected with Sec-pp65-A67-ACUAG RNA, Sec-pp65-2BgUTR-A120 RNA, orcontrol RNA (data not shown) or pulsed with pp65 peptide pool (1.75µg/ml) as positive control. After expansion for 7 days, each effectorcell population (4 × 10⁴/well) was assayed in an IFN-γ-ELISpot withautologous DCs (3 × 10⁴/well) which had been loaded either with pp65peptide pool or an irrelevant peptide pool (1.75 µg/ml). The graphicrepresentation depicts the average number of spots of triplicatemeasurements ± SEM.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

According to the invention, standard methods may be used for preparingrecombinant nucleic acids, culturing cells and introducing nucleicacids, in particular RNA, into cells, in particular electroporation andlipofection. Enzymatic reactions are carried out according to themanufacturers’ instructions or in a manner known per se.

According to the invention, a nucleic acid molecule or a nucleic acidsequence refers to a nucleic acid which is preferably deoxyribonucleicacid (DNA) or ribonucleic acid (RNA). According to the invention,nucleic acids comprise genomic DNA, cDNA, mRNA, recombinantly preparedand chemically synthesized molecules. According to the invention, anucleic acid may be in the form of a single-stranded or double-strandedand linear or covalently closed circular molecule.

“mRNA” means “messenger RNA” and refers to a “transcript” which isproduced using DNA as template and which itself codes for a peptide orprotein. An mRNA typically comprises a 5′-untranslated region, aprotein-encoding region and a 3′-untranslated region. mRNA has a limitedhalf time both in cells and in vitro. According to the invention, mRNAmay be prepared from a DNA template by in vitro transcription. It may bemodified by further stabilizing modifications and capping, in additionto the modifications according to the invention.

The term “nucleic acid” furthermore also comprises a chemicalderivatization of a nucleic acid on a nucleotide base, on the sugar oron the phosphate, and nucleic acids containing non-natural nucleotidesand nucleotide analogs.

According to the invention, a “nucleic acid sequence which is derivedfrom a nucleic acid sequence” refers to a nucleic acid containing, incomparison with the nucleic acid from which it is derived, single ormultiple nucleotide substitutions, deletions and/or additions and whichis preferably complementary to the nucleic acid from which it isderived, i.e. there is a certain degree of homology between said nucleicacids and the nucleotide sequences of said nucleic acids correspond in asignificant direct or complementary manner. According to the invention,a nucleic acid derived from a nucleic acid has a functional property ofthe nucleic acid from which it is derived. Such functional propertiesinclude in particular the ability to increase, in a functional linkageto a nucleic acid which can be transcribed into RNA (transcribablenucleic acid sequence), the stability and/or translation efficiency ofRNA produced from this nucleic acid in the complete RNA molecule.

According to the invention, “functional linkage” or “functionallylinked” relates to a connection within a functional relationship. Anucleic acid is “functionally linked” if it is functionally related toanother nucleic acid sequence. For example, a promoter is functionallylinked to a coding sequence if it influences transcription of saidcoding sequence. Functionally linked nucleic acids are typicallyadjacent to one another, where appropriate separated by further nucleicacid sequences, and, in particular embodiments, are transcribed by RNApolymerase to give a single RNA molecule (common transcript).

The nucleic acids described according to the invention are preferablyisolated. The term “isolated nucleic acid” means according to theinvention that the nucleic acid has been (i) amplified in vitro, forexample by polymerase chain reaction (PCR), (ii) recombinantly producedby cloning, (iii) purified, for example by cleavage andgel-electrophoretic fractionation, or (iv) synthesized, for example bychemical synthesis. An isolated nucleic acid is a nucleic acid availableto manipulation by recombinant DNA techniques.

A nucleic acid is “complementary” to another nucleic acid if the twosequences can hybridize with one another and form a stable duplex, saidhybridization being carried out preferably under conditions which allowspecific hybridization between polynucleotides (stringent conditions).Stringent conditions are described, for example, in Molecular Cloning: ALaboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, New York 1989 or CurrentProtocols in Molecular Biology, F.M. Ausubel et al., eds., John Wiley &Sons, Inc., New York, and refer, for example, to a hybridization at 65°C. in hybridization buffer (3.5 × SSC, 0.02% Ficoll, 0.02%polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH₂PO₄ (pH7),0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride/0.15 M sodiumcitrate, pH 7. After hybridization, the membrane to which the DNA hasbeen transferred, is washed, for example, in 2 × SSC at room temperatureand then in 0.1 - 0.5 × SSC/0.1 × SDS at temperatures up to 68° C.

According to the invention, complementary nucleic acids have nucleotideswhich are at least 60%, at least 70%, at least 80%, at least 90%, andpreferably at least 95%, at least 98% or at least 99%, identical.

The term “% identical” is intended to refer to a percentage ofnucleotides which are identical in an optimal alignment between twosequences to be compared, with said percentage being purely statistical,and the differences between the two sequences may be randomlydistributed over the entire length of the sequence and the sequence tobe compared may comprise additions or deletions in comparison with thereference sequence, in order to obtain optimal alignment between twosequences. Comparisons of two sequences are usually carried out bycomparing said sequences, after optimal alignment, with respect to asegment or “window of comparison”, in order to identify local regions ofcorresponding sequences. The optimal alignment for a comparison may becarried out manually or with the aid of the local homology algorithm bySmith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of thelocal homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol.48, 443, and with the aid of the similarity search algorithm by Pearsonand Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444 or with the aid ofcomputer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P,BLAST N and TFASTA in Wisconsin Genetics Software Package, GeneticsComputer Group, 575 Science Drive, Madison, Wis.).

Percentage identity is obtained by determining the number of identicalpositions in which the sequences to be compared correspond, dividingthis number by the number of positions compared and multiplying thisresult by 100.

For example, the BLAST program “BLAST 2 sequences” which is available onthe website http://www.ncbi.nlm.nih.gov/blast/b12seq/wblast2.cgi may beused.

“3′ end of a nucleic acid” refers according to the invention to that endwhich has a free hydroxy group. In a diagrammatic representation ofdouble-stranded nucleic acids, in particular DNA, the 3′ end is alwayson the right-hand side. “5′ end of a nucleic acid” refers according tothe invention to that end which has a free phosphate group. In adiagrammatic representation of double-strand nucleic acids, inparticular DNA, the 5′ end is always on the left-hand side.

In particular embodiments, a nucleic acid is functionally linkedaccording to the invention to expression control sequences which may behomologous or heterologous with respect to the nucleic acid.

A transcribable nucleic acid, in particular a nucleic acid coding for apeptide or protein, and an expression control sequence are“functionally” linked to one another, if they are covalently linked toone another in such a way that transcription or expression of thetranscribable and in particular coding nucleic acid is under the controlor under the influence of the expression control sequence. If thenucleic acid is to be translated into a functional peptide or protein,induction of an expression control sequence functionally linked to thecoding sequence results in transcription of said coding sequence,without causing a frame shift in the coding sequence or the codingsequence being unable to be translated into the desired peptide orprotein.

The term “expression control sequence” comprises according to theinvention promoters, ribosome-binding sequences and other controlelements which control transcription of a gene or translation of thederived RNA. In particular embodiments of the invention, the expressioncontrol sequences can be regulated. The precise structure of expressioncontrol sequences may vary depending on the species or cell type butusually includes 5′-untranscribed and 5′- and 3′-untranslated sequencesinvolved in initiating transcription and translation, respectively, suchas TATA box, capping sequence, CAAT sequence and the like. Morespecifically, 5′-untranscribed expression control sequences include apromoter region which encompasses a promoter sequence for transcriptioncontrol of the functionally linked gene. Expression control sequencesmay also include enhancer sequences or upstream activator sequences.

The nucleic acids specified herein, in particular transcribable andcoding nucleic acids, may be combined with any expression controlsequences, in particular promoters, which may be homologous orheterologous to said nucleic acids, with the term “homologous” referringto the fact that a nucleic acid is also functionally linked naturally tothe expression control sequence, and the term “heterologous” referringto the fact that a nucleic acid is not naturally functionally linked tothe expression control sequence.

The term “promoter” or “promoter region” refers to a DNA sequenceupstream (5′) of the coding sequence of a gene, which controlsexpression of said coding sequence by providing a recognition andbinding site for RNA polymerase. The promoter region may include furtherrecognition or binding sites for further factors involved in regulatingtranscription of said gene. A promoter may control transcription of aprokaryotic or eukaryotic gene. A promoter may be “inducible” andinitiate transcription in response to an inducer, or may be“constitutive” if transcription is not controlled by an inducer. Aninducible promoter is expressed only to a very small extent or not atall, if an inducer is absent. In the presence of the inducer, the geneis “switched on” or the level of transcription is increased. This isusually mediated by binding of a specific transcription factor.

Examples of promoters preferred according to the invention are promotersfor SP6, T3 or T7 polymerase.

According to the invention, the term “expression” is used in its mostgeneral meaning and comprises production of RNA or of RNA and protein.It also comprises partial expression of nucleic acids. Furthermore,expression may be transient or stable. With respect to RNA, the term“expression” or “translation” refers in particular to production ofpeptides or proteins.

The term “nucleic acids which can be transcribed to give a commontranscript” means that said nucleic acids are functionally linked to oneanother in such a way that, where appropriate after linearization suchas restriction enzyme cleavage of the nucleic acid molecule comprisingsaid nucleic acids, in particular of a closed circular nucleic acidmolecule, transcription under the control of a promoter results in anRNA molecule comprising the transcripts of said nucleic acids covalentlybound to one another, where appropriate separated by sequences locatedinbetween.

According to the invention, the term “transcription” comprises “in vitrotranscription”, wherein the term “in vitro transcription” relates to amethod in which RNA, in particular mRNA, is synthesized in vitro in acell-free manner, i.e. preferably by using appropriately prepared cellextracts. The preparation of transcripts preferably makes use of cloningvectors which are generally referred to as transcription vectors andwhich are included according to the invention under the term “vector”.

The term “nucleic acid sequence transcribed from a nucleic acidsequence” refers to RNA, where appropriate as part of a complete RNAmolecule, which is a transcription product of the latter nucleic acidsequence.

The term “nucleic acid sequence which is active in order to increase thetranslation efficiency and/or stability of a nucleic acid sequence”means that the first nucleic acid is capable of modifying, in a commontranscript with the second nucleic acid, the translation efficiencyand/or stability of said second nucleic acid in such a way that saidtranslation efficiency and/or stability is increased in comparison withthe translation efficiency and/or stability of said second nucleic acidwithout said first nucleic acid. In this context, the term “translationefficiency” relates to the amount of translation product provided by anRNA molecule within a particular period of time and the term “stability”relates to the half life of an RNA molecule.

The 3′-untranslated region relates to a region which is located at the3′ end of a gene, downstream of the termination codon of aprotein-encoding region, and which is transcribed but is not translatedinto an amino acid sequence.

According to the invention, a first polynucleotide region is consideredto be located downstream of a second polynucleotide region, if the 5′end of said first polynucleotide region is the part of said firstpolynucleotide region closest to the 3′ end of said secondpolynucleotide region.

The 3′-untranslated region typically extends from the termination codonfor a translation product to the poly(A) sequence which is usuallyattached after the transcription process. The 3′-untranslated regions ofmammalian mRNA typically have a homology region known as the AAUAAAhexanucleotide sequence. This sequence is presumably the poly(A)attachment signal and is frequently located from 10 to 30 bases upstreamof the poly(A) attachment site.

3′-untranslated regions may contain one or more inverted repeats whichcan fold to give stem-loop structures which act as barriers forexoribonucleases or interact with proteins known to increase RNAstability (e.g. RNA-binding proteins).

5′- and/or 3′-untranslated regions may, according to the invention, befunctionally linked to a transcribable and in particular coding nucleicacid, so as for these regions to be associated with the nucleic acid insuch a way that the stability and/or translation efficiency of the RNAtranscribed from said transcribable nucleic acid are increased.

The 3′-untranslated regions of immunoglobulin mRNAs are relatively short(fewer than about 300 nucleotides), while the 3′-untranslated regions ofother genes are relatively long. For example, the 3′-untranslated regionof tPA is about 800 nucleotides in length, that of factor VIII is about1800 nucleotides in length and that of erythropoietin is about 560nucleotides in length.

It can be determined according to the invention, whether a3′-untranslated region or a nucleic acid sequence derived therefromincreases the stability and/or translation efficiency of RNA, byincorporating the 3′-untranslated region or the nucleic acid sequencederived therefrom into the 3′-untranslated region of a gene andmeasuring whether said incorporation increases the amount of proteinsynthesized.

The above applies accordingly to the case in which according to theinvention a nucleic acid comprises two or more 3′-untranslated regionswhich are preferably coupled sequentially with or without a linkerinbetween, preferably in a “head-to-tail relationship” (i.e. the3′-untranslated regions have the same orientation, preferably theorientation naturally occurring in a nucleic acid).

According to the invention, the term “gene” refers to a particularnucleic acid sequence which is responsible for producing one or morecellular products and/or for achieving one or more intercellular orintracellular functions. More specifically, said term relates to a DNAsection which comprises a nucleic acid coding for a specific protein ora functional or structural RNA molecule.

The terms “polyadenyl cassette” or “poly(A) sequence” refer to asequence of adenyl residues which is typically located at the 3′ end ofan RNA molecule. The invention provides for such a sequence to beattached during RNA transcription by way of a DNA template on the basisof repeated thymidyl residues in the strand complementary to the codingstrand, whereas said sequence is normally not encoded in the DNA but isattached to the free 3′ end of the RNA by a template-independent RNApolymerase after transcription in the nucleus. According to theinvention, a poly(A) sequence of this kind is understood as meaning anucleotide sequence of at least 20, preferably at least 40, preferablyat least 80, preferably at least 100 and preferably up to 500,preferably up to 400, preferably up to 300, preferably up to 200, and inparticular up to 150, consecutive A nucleotides, and in particular about120 consecutive A nucleotides, wherein the term “A nucleotides” refersto adenyl residues.

In a preferred embodiment, a nucleic acid molecule according to theinvention is a vector. The term “vector” is used here in its mostgeneral meaning and comprises any intermediate vehicles for a nucleicacid which, for example, enable said nucleic acid to be introduced intoprokaryotic and/or eukaryotic host cells and, where appropriate, to beintegrated into a genome. Such vectors are preferably replicated and/orexpressed in the cell. Vectors comprise plasmids, phagemids or virusgenomes. The term “plasmid”, as used herein, generally relates to aconstruct of extrachromosomal genetic material, usually a circular DNAduplex, which can replicate independently of chromosomal DNA.

According to the invention, the term “host cell” refers to any cellwhich can be transformed or transfected with an exogenous nucleic acid.The term “host cell” comprises, according to the invention, prokaryotic(e.g. E. coli) or eukaryotic cells (e.g. yeast cells and insect cells).Particular preference is given to mammalian cells such as cells fromhumans, mice, hamsters, pigs, goats, primates. The cells may be derivedfrom a multiplicity of tissue types and comprise primary cells and celllines. Specific examples include keratinocytes, peripheral bloodleukocytes, bone marrow stem cells and embryonic stem cells. In otherembodiments, the host cell is an antigen-presenting cell, in particulara dendritic cell, a monocyte or a macrophage. A nucleic acid may bepresent in the host cell in a single or in several copies and, in oneembodiment is expressed in the host cell.

According to the invention, a peptide or protein encoded by a nucleicacid may be a peptide or protein which is located in the cytoplasma, inthe nucleus, in the membrane, in organelles or is secreted. They includestructural proteins, regulatory proteins, hormones, neurotransmitters,growth-regulating factors, differentiation factors, geneexpression-regulating factors, DNA-associated proteins, enzymes, serumproteins, receptors, medicaments, immunomodulators, oncogenes, toxins,tumor antigens or antigens. Said peptides or proteins may have anaturally occurring sequence or a mutated sequence in order to enhance,inhibit, regulate or eliminate their biological activity.

The term “peptide” refers to substances which comprise two or more,preferably 3 or more, preferably 4 or more, preferably 6 or more,preferably 8 or more, preferably 10 or more, preferably 13 or more,preferably 16 or more, preferably 20 or more, and up to preferably 50,preferably 100 or preferably 150, consecutive amino acids linked to oneanother via peptide bonds. The term “protein” refers to large peptides,preferably peptides having at least 151 amino acids, but the terms“peptide” and “protein” are used herein usually as synonyms. The terms“peptide” and “protein” comprise according to the invention substanceswhich contain not only amino acid components but also non-amino acidcomponents such as sugars and phosphate structures, and also comprisesubstances containing bonds such as ester, thioether or disulfide bonds.

The invention provides for nucleic acids, in particular RNA, to beadministered to a patient. In one embodiment, nucleic acids areadministered by ex vivo methods, i.e. by removing cells from a patient,genetically modifying said cells and reintroducing the modified cellsinto the patient. Transfection and transduction methods are known to theskilled worker. The invention also provides for nucleic acids to beadministered in vivo.

According to the invention, the term “transfection” refers tointroducing one or more nucleic acids into an organism or into a hostcell. Various methods may be employed in order to introduce according tothe invention nucleic acids into cells in vitro or in vivo. Such methodsinclude transfection of nucleic acid-CaPO₄ precipitates, transfection ofnucleic acids associated with DEAE, transfection or infection withviruses carrying the nucleic acids of interest, liposome-mediatedtransfection, and the like. In particular embodiments, preference isgiven to directing the nucleic acid to particular cells. In suchembodiments, a carrier used for administering a nucleic acid to a cell(e.g. a retrovirus or a liposome) may have a bound targeting molecule.For example, a molecule such as an antibody specific to a surfacemembrane protein on the targeted cell, or a ligand for a receptor on thetarget cell may be incorporated into or bound to the nucleic acidcarrier. If administration of a nucleic acid by liposomes is desired,proteins binding to a surface membrane protein associated withendocytosis may be incorporated into the liposome formulation in orderto enable targeting and/or absorption. Such proteins include capsidproteins or fragments thereof which are specific to a particular celltype, antibodies to proteins that are internalized, proteins targetingan intracellular site, and the like.

“Reporter” relates to a molecule, typically a peptide or protein, whichis encoded by a reporter gene and measured in a reporter assay.Conventional systems usually employ an enzymatic reporter and measurethe activity of said reporter.

The term “multiple cloning site” refers to a nucleic acid regioncontaining restriction enzyme sites, any one of which may be used forcleavage of, for example, a vector and insertion of a nucleic acid.

According to the invention, two elements such as nucleotides or aminoacids are consecutive, if they are directly adjacent to one another,without any interruption. For example, a sequence of x consecutivenucleotides N refers to the sequence (N)_(x).

“Restriction endonuclease” or “restriction enzyme” refers to a class ofenzymes that cleave phosphodiester bonds in both strands of a DNAmolecule within specific base sequences. They recognize specific bindingsites, referred to as recognition sequences, on a double-stranded DNAmolecule. The sites at which said phosphodiester bonds in the DNA arecleaved by said enzymes are referred to as cleavage sites. In the caseof type IIS enzymes, the cleavage site is located at a defined distancefrom the DNA binding site. According to the invention, the term“restriction endonuclease” comprises, for example, the enzymes SapI,EciI, BpiI, AarI, AloI, BaeI, BbvCI, PpiI and PsrI, BsrD1, BtsI, Earl,BmrI, BsaI, BsmBI, FauI, BbsI, BciVI, BfuAI, BspMI, BseRI, EciI, BtgZI,BpuEI, BsgI, Mmel, CspCI, BaeI, BsaMI, Mva1269I, PctI, Bse3DI, BseMI,Bst6I, Eam1104I, Ksp632I, BfiI, Bso31I, BspTNI, Eco31I, Esp3I, BfuI,Acc36I, AarI, Eco57I, Eco57MI, GsuI, AloI, Hin4I, PpiI, and PsrI.

“Half life” refers to the time required for eliminating half of theactivity, amount or number of molecules.

The present invention is described in detail by the following figuresand examples which should be construed by way of illustration only andnot by way of limitation. On the basis of the description and theexamples, further embodiments are accessible to the skilled worker andare likewise within the scope of the invention.

EXAMPLES Example 1: Preparation of Vectors and In Vitro Transcription ofRNA

In order to study the effects of the RNA modifications according to theinvention on the level and duration of expression, a number of IVTvectors were prepared which served as template for in vitrotranscription (FIG. 3 ).

The reporter genes for eGFP and d2eGFP, two molecules with differenthalf lives (HL), were inserted into the vectors, thereby enabling theinfluence of the RNA modifications according to the invention to beanalyzed. Fluorescence decreases with an average HL of 17.3 h for eGFPand 2 h for d2eGFP. These constructs were used for preparing invitro-transcribed eGFP RNA and d2eGFP RNA, respectively.

Example 2: Transfection of Cells With the In Vitro-Transcribed RNAModified According to the Invention and Effect on RNA Translation andStability

In vitro-transcribed eGFP RNA and d2eGFP RNA were used for transfectingK562 cells (human, leukemia) by means of electroporation. Thetransfection efficiency was > 90% in K562 cells.

This was followed by assaying the action of the RNA modificationsdescribed in human dendritic cells (DCs) which are the most importantmodulators of the immune system. This approach is immunologicallyrelevant because RNA-transfected DCs can be considered for vaccination.Immature DCs are located in the skin and in peripheral organs. Here theyare in an immature state which is characterized by well-studied surfacemarkers and which is functionally distinguished by high endocytoticactivity. An immunogenic stimulus such as, for example, an infectionwith pathogens, triggers a DC maturation process. At the same time, saidstimulus initiates DC migration into the regional lymph nodes, wheresaid DCs are the most effective inducers of T cell and B cell immuneresponses. The mature state of said DCs is also characterized byexpression of surface markers and cytokines studied in detail and by acharacteristic DC morphology. There are established cell culture systemsfor differentiating immature human DCs from blood monocytes. These maybe caused to mature by various stimuli.

The transfection efficiency in primary dendritic cells was 70-80%. TheDCs were stained with anti-CD80, anti-CD83, anti-CD86 and anti-HLA-DRantibodies which recognize specific DC maturation markers, and analyzedby flow cytometry (FIG. 4 ).

The level and duration of expression were determined with the aid ofFACS-Kalibur by way of determining the eGFP fluorescence intensity. Theamount of RNA in the cells was determined with the aid of a quantitativeRT-PCR.

A. Effect of an Open-ended poly(A) Sequence on RNA Translation andStability

Both the tumor cell line K562 and immature DCs (iDC) were shown totranslate RNA having an open-ended poly(A) sequence more efficiently andover a longer period of time than RNA having a masked-end poly(A)sequence (FIG. 5 a ). The translation efficiency for an unmasked-endpoly(A) sequence in immature DCs is increased by a factor of 1.5, withpoly(A) sequences of equal length. Moreover, said modification resultsin higher RNA stability (FIG. 5 b ). A 4 to 5-fold amount of RNA can bedetected in immature DCs which had been transfected with RNA having anunmasked poly(A) sequence 48 h after electroporation.

B. Effect of the poly(A) Sequence Length on RNA Translation andStability

The analysis of RNA having poly(A) sequences of 16bp, 42bp, 51bp, 67bp,120bp, 200bp, 300bp and 600bp in length revealed that extension of saidpoly(A) sequence up to 120 A nucleotides increases transcript stabilityand translation and that an extension going beyond that has no positiveeffect. This effect is observed both in K562 cells and in immature DCs(iDC) (FIGS. 6 a and 6 b ). Extending the poly(A) sequence from 51 to120 A nucleotides produces a 1.5 to 2-fold increase in translationefficiency. This effect is also reflected in RNA stability (FIG. 6 c ).

C. Effect of the Occurrence of a 3′-Untranslated Region on RNATranslation and Stability

A time course with K562 cells and immature DCs confirmed thatintroducing a 3′-untranslated region (UTR) of human beta-globin resultsin increasing expression of an RNA transcript. In addition, it wasdemonstrated that a double 3′-untranslated region (UTR) of humanbeta-globin results in an enhanced level of expression after 24 h, whichmarkedly exceeds the combined effect of two individual UTRs (FIG. 7 ).

D. Effect of a Combination of the Above-Described Modifications on RNATranslation and Stability

According to the invention, a combination of the above-describedmodifications in an RNA transcript was shown to increase the translationefficiency of eGFP in immature and also in mature DCs by a factor ofgreater than five (FIG. 8 ).

Example 3: Presentation of a Peptide Expressed via In Vitro-TranscribedRNA with Increased Stability and Translation Efficiency by MHC Molecules

According to the invention, the use of RNA constructs modified accordingto the invention was shown to increase peptide-MHC presentation on thecell surface. To this end, the nucleic acid sequence coding for eGFP inthe IVT vectors described was replaced with a nucleic acid sequencecoding for the OVA257-264 epitope (SIINFEKL), and the constructs werecompared with one another. The target cells used for transfection wereEL4 cells (murine, T cell lymphoma).

In order to quantify SIINFEKL peptides presented by MHC molecules, thecells were stained with an anti-H2-K^(b)-OVA257-264 antibody at varioustime points after electroporation, and the fluorescence intensity of asecondary antibody was determined with the aid of FACS-Kalibur (FIG. 9).

Furthermore, the SIINFEKL peptide was cloned into the vector whichreflected all optimizations (pST1-Sec-SIINFEKL-2BgUTR-A120-Sap1) andinto a vector with standard features (pST1-Sec-SIINFEKL-A67-Spel). IVTRNA derived from both vectors was electroporated into EL4 cells andBMDCs. OVA-peptide/K^(b) complexes were found on the cell surface insubstantially greater numbers and were maintained over a longer periodof time after electroporation of the RNA modified according to theinvention, Sec-SIINFEKL-2-BgUTR-A120 (FIG. 10 ).

Example 4: Effect of a Transfection of Cells With In Vitro-TranscribedRNA Coding for a Peptide to be Presented on the Expansion ofAntigen-Specific T Cells

In order to evaluate the effect on stimulatory capacity, OT-I-TCR wasemployed which had been used intensively in the C57BL/J6 (B6) backgroundin order to detect MHC class I presentation of the SIINFEKL peptide.OT-I CD8⁺ T cells which are transgenic with regard to the T cellreceptor (TCR) and which recognize the K^(b)-specific peptide SIINFEKLfrom chicken OVA (OVA₂₅₇₋₂₆₄), was kindly provided by H. Schild(Institute of Immunology, University of Mainz, Germany).

On day 0, animals underwent adoptive transfer with OT-I-CD8⁺ T cells. Tothis end, splenocytes were prepared from TCR tg OT-I mice and introducedinto the tail vein of C57BL/J6 recipient mice. The cell number wasadjusted to 1 × 10⁵ TCR tg CD8⁺ T cells. On the next day, 1 × 10⁶ BMDCsof C57BL/J6 mice which had been electroporated with 50 pmol ofSIINFEKL-encoding RNA construct variants and had been allowed to matureby means of poly(I:C) for 16 hours were administered ip to mice. On day4, OT-I-CD8⁺ T cells were measured in peripheral blood with the aid ofthe tetramer technology. To this end, retroorbital blood samples weretaken and stained with anti-CD8 (Caltag Laboratories, Burlingame, USA)and SIINFEKL tetramer (H-2Kb/SIINFEKL 257-264; Beckman Coulter,Fullerton, USA) .

In vivo expansion of antigen-specific TCR-transgenic CD8⁺ T cells wasfound to be substantially improved when using Sec-SIINFEKL-2BgUTR-A120RNA for antigen supply in comparison with Sec-SIINFEKL-A67-ACUAG RNA(FIG. 11A).

In order to evaluate, whether stabilized IVT RNA constructs for antigensupply also improve antigen-specific stimulation of human T cells,HCMV-pp65, the immunodominant antigen of human cytomegalovirus which isoften used for validating autologous stimulation of polyepitopic T cellreactions, was employed. CD4⁺ and CD8⁺ T cells which had been purifiedfrom HCMV-seropositive healthy donors by positive magnetic cell sortingby means of antibody-coated microbeads (Miltenyi Biotec,Bergisch-Gladbach, Germany) were cocultured with 2 × 10⁵ autologous DCswhich had been electroporated with the corresponding IVT RNA variantscoding for pp65. An expansion of T cells, measured on day 7 in anIFN-γ-ELISpot using autologous DCs which had been pulsed with a pool ofoverlapping peptides covering the entire pp65 protein sequence, or witha control protein, demonstrated the superiority of Sec-pp65-2BgUTR-A120,with the effects with regard to expansion of CD4⁺ T cells being the mostpronounced (FIG. 11B).

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1 -
 66. (canceled)
 67. An immunogenic composition comprising: an RNAmolecule comprising, in a 5′ to 3′ orientation: (a) a first nucleic acidsequence comprising a 5′-untranslated region (5′-UTR), (b) a secondnucleic acid sequence comprising a sequence that encodes an antigen, and(c) a third nucleic acid sequence comprising a 3′-terminal poly(A) tailsequence of about 100 to about 150 consecutive A nucleotides, whereinthe 3′-terminal nucleotide of the RNA molecule is an A nucleotide;wherein the 5′-UTR is heterologous with respect to the sequence thatencodes the antigen.
 68. The immunogenic composition of claim 67,wherein the third nucleic acid sequence (c) is active so as to increasetranslation efficiency and/or stability of the RNA molecule.
 69. Theimmunogenic composition of claim 67, wherein the 3′ terminal poly(A)tail sequence has a length of about 100 consecutive A nucleotides. 70.The immunogenic composition of claim 67, wherein the 3′ terminal poly(A)tail sequence has a length of about 120 consecutive A nucleotides. 71.The immunogenic composition of claim 67, wherein the RNA moleculecomprises a 5′ cap.
 72. The immunogenic composition of claim 67, whereinthe 5′-UTR is or comprises a 5′ UTR of a globin gene.
 73. Theimmunogenic composition of claim 67, wherein the 5′-UTR comprises aribosome binding sequence.
 74. The immunogenic composition of claim 67,wherein the RNA molecule is an mRNA molecule.
 75. The immunogeniccomposition of claim 67, wherein the composition is a vaccine.
 76. Theimmunogenic composition of claim 67, wherein the RNA molecule comprisesa non-natural nucleotide analog.
 77. The immunogenic composition ofclaim 67, wherein the antigen is or comprises a tumor antigen.
 78. Theimmunogenic composition of claim 67, wherein the RNA molecule is an invitro transcribed RNA molecule.
 79. A mammalian cell comprising: an RNAmolecule comprising, in a 5′ to 3′ orientation: (a) a first nucleic acidsequence comprising a 5′-untranslated region (5′-UTR), (b) a secondnucleic acid sequence comprising a sequence that encodes a peptide or aprotein, and (c) a third nucleic acid sequence comprising a 3′-terminalpoly(A) tail sequence of about 100 to about 150 consecutive Anucleotides, wherein the 3′-terminal nucleotide of the RNA molecule isan A nucleotide; wherein the 5′-UTR is heterologous with respect to thesequence that encodes the peptide or the protein; and wherein expressionand/or stability of the RNA molecule in the cell is increased relativeto that as observed with an RNA molecule with a poly(A) tail sequence ofno more than 51 consecutive A nucleotides.
 80. The mammalian cell ofclaim 79, wherein expression and/or stability of the RNA molecule isincreased relative to that as observed with an RNA molecule with apoly(A) tail sequence of about 51 consecutive A nucleotides.
 81. Themammalian cell of claim 79, wherein the 3′ terminal poly(A) tailsequence has a length of about 100 consecutive A nucleotides.
 82. Themammalian cell of claim 79, wherein the RNA molecule comprises at leastone of the following features: a 5′ cap; a non-natural nucleotideanalog; a 5′ UTR that is or comprises a 5′ UTR of a globin gene; and a5′ UTR that is or comprises a ribosome binding sequence.
 83. Themammalian cell of claim 79, wherein the RNA molecule is an in vitrotranscribed RNA molecule, and/or is an mRNA molecule.
 84. The mammaliancell of claim 79, wherein the peptide or protein is or comprises anantigen.
 85. An antigen-presenting cell comprising: an mRNA moleculecomprising, in a 5′ to 3′ orientation: (a) a first nucleic acid sequencecomprising a 5′-untranslated region (5′-UTR), (b) a second nucleic acidsequence comprising a sequence that encodes a peptide or a protein, and(c) a third nucleic acid sequence comprising a 3′-terminal poly(A) tailsequence of about 100 to about 150 consecutive A nucleotides, whereinthe 3′-terminal nucleotide of the RNA molecule is an A nucleotide;wherein the 5′-UTR is heterologous with respect to the sequence encodingthe peptide or the protein.
 86. The antigen-presenting cell of claim 85,wherein the antigen-presenting cell is a dendritic cell.
 87. Theantigen-presenting cell of claim 85, wherein the peptide or the proteinis or comprises an antigen.
 88. The antigen-presenting cell of claim 85,wherein the cell is present in a subject administered with the mRNAmolecule.
 89. The antigen-presenting cell of claim 85, wherein the 3′terminal poly(A) tail sequence has a length of about 100 consecutive Anucleotides.
 90. The antigen-presenting cell of claim 85, wherein themRNA molecule comprises at least one of the following features: a 5′cap; a non-natural nucleotide analog; a 5′ UTR that is or comprises a 5′UTR of a globin gene; and a 5′ UTR that is or comprises a ribosomebinding sequence.
 91. The antigen-presenting cell of claim 85, whereinthe mRNA molecule is an in vitro transcribed mRNA molecule.
 92. Theantigen-presenting cell of claim 85, wherein expression and/or stabilityof the mRNA molecule is increased relative to that as observed with anmRNA molecule with a poly(A) tail sequence of about 51 consecutive Anucleotides.
 93. A method for stimulating or expanding antigen-specificT cells, the method comprising steps of: (a) obtaining an mRNA moleculecomprising: (i) a coding sequence that encodes an antigen or a peptideor a protein comprising the antigen, and (ii) a 3′-terminal poly(A) tailsequence of about 100 to about 150 consecutive A nucleotides, whereinthe 3′-terminal nucleotide of the mRNA molecule is an A nucleotide; (b)delivering the mRNA molecule to antigen-presenting cells underconditions such that the encoded antigen or the encoded peptide orpolypeptide is expressed in the antigen-presenting cells; and (c)exposing T cells to the antigen presenting cells from (b) underconditions that stimulates or expands antigen-specific T cells, whereinthe number of antigen-specific T cells is increased, relative to that asobserved with an mRNA molecule having a poly(A) tail sequence of no morethan 67 consecutive A nucleotides.
 94. The method of claim 93, whereinthe T cells are or comprise CD4⁺ and/or CD8⁺ T cells, and/or wherein theantigen-presenting cells are or comprise dendritic cells.
 95. The methodof claim 93, wherein the mRNA molecule is delivered to theantigen-presenting cells by transfection in vitro.
 96. The method ofclaim 93, wherein expression and/or stability of the mRNA molecule isincreased relative to that as observed with an mRNA molecule with apoly(A) tail sequence of about 51 consecutive A nucleotides.